| Objective:To study the effects of Di(2-ethylhexyl)phthalate(DEHP)and its metabolite mono(2-ethylhexyl)phthalate(MEHP)on HepG2 cells;to further explore the lipid metabolism in liver injury.Methods:HepG2 cells were respectively treated with the DEHP(31.25,62.5,125.0,250.0,500.0,1000.0 μM)and the MEHP(3.125,6.25,12.5,25.0,50.0,100.0,250.0 μM).Negative control was treated with DMEM,After 24 h,48 h and 72 h treatment,the cells viability was detected by MTT assay.The HepG2 cells were respectively treated with the DEHP(10.0,250.0 μM)and MEHP(4.0,100.0 μM),negative control(DMEM),positive controls were treated with 0.5 mM oleic acid(OA)and 0.1% DMSO(dimethyl sulfoxide)for 48 h,then stained with oil red O staining.The HepG2 cells were respectively treated with DEHP(2.0,10.0,50.0,250.0 μM),MEHP(0.8,4.0,20.0 and 100.0 μM),0.5 mM OA and 0.5 μg/ml BFA for 24 h and 48 h,the intra and extra-hepatocellular triglyceride(TG)contents were assessed.ELISA was used to evaluate soluble Malonyl-CoA(mCoA)expression levels;western blot was applied to detect the expressions of TG related proteins AMP-activated protein kinase(AMPKα)、Phospho-AMP-activated protein kinase(p-AMPK)、 Acetyl-CoA carboxylase(ACC1)、 Phospho-Acetyl-CoA carboxylase(p-ACC1)、 Sterol regulatory element binding protein 1c(SREBP-1c)、 Carnitine palmitoyltransferase 1A(CPT1A)、Malonyl-CoA deearboxylase(MLYCD)、 Adipose triglyceride lipase(ATGL)、Silent information regulator 1(SIRT1)、 Peroxisome proliferator-activated receptor α(PPARα)、 p53 tumor suppressor protein(p53)、 phosphorylated p53 tumor suppressor protein(p-p53),adipose tissue triglyceride hydrolase(ATGL)and microsomal triglyceride transfer protein(MTP).Results:1.DEHP treatment(500.0~1000.0 μM,24 /48 /72 h)significantly supressed the viability of HepG2 cells(P<0.05).MEHP treatment(50.0~250.0 μM,24 /48 h)and 250.0 μM MEHP treatment 72 h significantly decreased cell viability(P <0.05).2.Compared with the negative control,the expression levels of malonyl-CoA in HepG2 cells were increased after treated with 50.0~250.0 μM DEHP and 0.8~100.0 μM MEHP for 24 h and 48 h,respectively,were significantly different(P <0.05).3.Compared with the negative control,50.0~250.0 μM DEHP exposure 48 h increased Hep G2 extracellular triglyceride levels;100.0 μM MEHP exposure 48 h increased HepG2 extracellular triglyceride levels.DEHP treatment(50.0~250.0 μM)for 24 h and 48 h increased intracellular TG level in the HepG2 cells;MEHP treatment(20.0~100.0 μM)for 24 h and 48 h significantly increased intracellular TG level in the hepatocytes(P <0.05).4.Oil red O staining showed that,after exposure 48 h,10.0~250.0 μM DEHP exposed groups and 4.0~100.0 μM MEHP groups in HepG2 cells exposed to visible lipid droplets,and accompanied by lipid droplets fusion phenomenon.5.Compared with the negative control,the expression of ACC1 protein was up-regulated in HepG2 cells treated with 2.0~250.0 μM DEHP and 20.0~100.0 μM MEHP for 24 h;however it was down-regulated when treated with 250.0 μM DEHP and 20.0~100.0 μM MEHP for 48 h.The expression of p-ACC protein was up-regulated in HepG2 cells by the treatments of 250.0 μM DEHP and 100.0 μM MEHP(24 h),50.0~250.0 μM DEHP and 20.0~100.0 μM MEHP(48 h).After(2.0~250.0 μM)DEHP and(0.8~100.0 μM)MEHP treatment for 24 h and 48 h,the expression of SREBP-1c protein was not changed.The expression of AMPK of HepG2 cells was similar after treated with DEHP(2.0~250.0 μM)and MEHP(0.8~100.0 μM)for 24 h and 48 h;the expression of p-AMPK was up-regulated in HepG2 cells treated with 50.0~250.0 μM DEHP and 0.8~100.0 μM MEHP for 48 h,but no difference was found after 24 h treatment.6.Compared with the negative control,the expression of ATGL in HepG2 cells was significantly down-regulated when treated with 250.0 μM DEHP and 20.0~100.0 μM MEHP for 24 h,10.0~250.0 μM DEHP and 4.0~100.0 μD MEHP for 48 h(P <0.05).7.Compared with the negative control,the expression of p53 protein in HepG2 cells was up-regulated after treated with 50.0~250.0 μM DEHP and 0.8~100.0 μM MEHP for 24 h,10.0~250.0 μM DEHP and 0.8~100.0 μM MEHP for 48 h;the expression of p-p53 protein was significantly increased in HepG2 cells after treated with 10.0~250.0 μM DEHP and 0.8~100.0 μM MEHP for 24 h and 48 h;the SIRT1 protein expression was greatly decreased after 50.0~250.0 μM DEHP treatment for 24 h and 48 h,the 100.0 μM MEHP treatment for 24 h and 20.0~100.0 μM MEHP treatment for 48 h decreased the SIRT1 protein expressions,the difference was significant(P <0.05).The expression of PPARα protein was down-regulated in HepG2 cells when treated with 2.0~250.0 μM DEHP and 0.8~100.0 μM MEHP for 24 h and 48 h;different dose of DEHP and MEHP treatment 24 h did not make the difference of MLYCD expression in HepG2 cells,after(10.0~250.0 μM)DEHP and(4.0~100.0 μM)MEHP treatment for 48 h,the expression of MLYCD protein was down-regulated;The level of CPT1 A expression was reduced by 250.0 μM DEHP,100.0 μM MEHP for 24 h,50.0~250.0 μM DEHP and 4.0~100.0 μM MEHP for 48 h.8.Compared with the negative control,the MTP expression in HepG2 cells was significantly decreased after treated with 250.0 μM DEHP and 20.0~100.0 μM MEHP for 48 h.After 24 h DEHP(2.0~250.0 μM)and MEHP treatment(0.8~100.0 μM),the expression of MTP protein remained unchanged.Conclusions: 1.High levels of DEHP and MEHP treatment could supress cell viability of hepatocytes.2.DEHP and MEHP exposure can affect the fat metabolism of HepG2 cells.3.DEHP and MEHP exposure can cause fat accumulation in HepG2 cells this process may caused by blocking of with β-oxidation of fatty acids and inhibition of triglyceride hydrolysis. |
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